Overcoming chromoprotein limitations by engineering a red fluorescent protein

Letian Bao,P Navaneeth K Menon,Josefine Liljeruhm,Anthony C Forster

doi:10.1016/j.ab.2020.113936

Abstract

Chromoproteins (CPs) are widely-used visual reporters of gene expression. We previously showed that, for coloration in Escherichia coli, CPs had to be overexpressed and that this caused large fitness costs with the most useful (darkly colored) CPs. These fitness costs were problematic because passage of plasmids encoding darkly colored CPs in liquid culture frequently resulted in loss of color due to mutations. Unexpectedly, an early variant of the monomeric red fluorescent protein 1 (mRFP1) gene that was codon-optimized for E. coli (abbreviated mRFP1E) was found here to be an ideal replacement for CP genes. When we subcloned mRFP1E in the same way as our CP genes, it produced a similarly dark color, yet affected E. coli fitness minimally. This finding facilitated testing of several hypotheses on the cause of CP cytotoxicities by gel electrophoresis and size-exclusion chromatography: toxicities correlated with the combination of amounts of expression, oligomerization and inclusion bodies, not isoelectric point. Finally, a semi-rational mutagenesis strategy created several mRFP1 protein variants with different colors without altering the fitness cost. Thus, these mutants and mRFP1E are suitable for comparative fitness costs between different strains of E. coli. We conclude that our new mRFP1E series overcomes prior limitations of CPs.

Highlights

The family of eukaryotic fluorescent proteins (FPs; [1]), chromo proteins (CPs; [2]) and their engineered variants are widely used as reporters for gene expression
BioBrick plasmid vectors and parts specified with BBa numbers below were obtained from the Registry of Standard Biological Parts. pSB1C3, pSB1K3, pSB3K3 and pSB1K3 contained the E. coli-codon-optimized, BioBrickrestriction-enzyme-free (RFC10 standard) monomeric red fluorescent protein 1 (mRFP1) BBa_E1010 marker gene under the control of a LacIrepressible promoter
We reasoned that several approaches might potentially yield a CP gene that could be overexpressed from a high-copy plasmid in E. coli to give a darkly colored CP with a low fitness cost

Summary

Introduction

The family of eukaryotic fluorescent proteins (FPs; [1]), chromo proteins (CPs; [2]) and their engineered variants are widely used as reporters for gene expression. They derive from jellyfish and corals, sharing a homologous three-amino-acid fluorophore/chromophore which self-matures post-translationally within a β-barrel by reacting with oxygen [1]. All of the really useful (darkly colored and fast-maturing) CPs often lost their colors in liquid culture, which was challenging for maintenance of stocks and suboptimal when choosing a reporter [9], especially for strain competition studies. Loss of CP expression was negligible after re-streaking on LB agar plates from fresh plates because solid culturing alone (with each colony starting from a single cell bottleneck) reduces bacterial and plasmid generations compared with solid liquid culturing (with each liquid culture starting from up to 108 cells/colony), and because better selection occurs in liquid culture due to free bacterial movement in three dimensions [10]

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Results

Conclusion